Novel genetic and epigenetic alterations in ... - Ous-research.no

Neoplasia Vol. 10, No. 7, 2008 RAS Signaling in Colorectal Carcinomas Ahlquist et al. 681oncogene since its first mutational report in 1984 [3], and it is nowknown that it is mutated in 21% of all human sporadic cancers, includingone third of CRCs [1]. 2 BRAF was shown to be a mutationaltarget in cancer 5 years ago [4], and 20% of all human cancers harborsa mutation, including an estimated 13% of colorectal carcinomas.2 Another potential target of this pathway is the NF1 gene,which encodes neurofibromatosis type 1, a GTPase-activating protein(GAP), governing hydrolysis of KRAS-GTP to KRAS-GDP[5], thereby functioning as a negative regulator of KRAS signaling.The NF1 gene is approximately 280 kb in size and maps to chromosome17q11.2. It contains 61 exons, with an 11- to 13-kb transcriptand an open reading frame coding for 2818 amino acids. There aretwo catalytical domains in NF1, which are important for its function,namely, the cAMP/PKA domain comprising exons 11 to 17 and theRAS-GRD (RAS GAP-related domain) domain comprising exons21 to 27a [6–8]. Neurofibromatosis type 1, a dominant disorder,is caused by mutations in NF1, but somatic mutations in this genecan also contribute to tumorigenesis. Since the first mutation reportof the gene in 1992 showing that one colorectal tumor (of 22) wasmutated in NF1 [9], it has been speculated to play a role in colorectaltumorigenesis. However, due to the large size of the gene and thefact that there are no mutational hot spots, mutation analysis ofNF1 in tumors has been very scarce. RASSF1 (Ras association domainfamily 1) gene maps at chromosome 3p21.3, and its isoformA(RASSF1A) has been found hypermethylated in 40% of lung tumors[10] and in a large variety of human cancers, including CRC[11,12]. As implied by its designation, RASSF1A is thought to interactwith KRAS through a Ras association domain that alters itseffects. RASSF1A has several effects, including promotion of apoptosis,cell cycle arrest, and maintenance of genomic stability, abilitiestypical of tumor suppressor genes. Some of these effects refer tothe negative regulation of KRAS [13]. Its association to, and its effecton, KRAS is still not solved, although increasing evidence points to adirect binding between RASSF1A and farnesylated KRAS (reviewedin the study of Donninger et al. [11]). The KRAS and BRAF mutationstatus together with the alteration of other upstream componentsaffecting the RAS signaling have been reported for othercancers [14], but only two previous studies have examined alterationsin KRAS, BRAF, and RASSF1A in the same series of colorectal neoplasms[15,16], and independent of cancer type, no previous studyhas included a detailed analyses of the NF1 gene.To provide further insight into the role of MAP kinase signaling inCRC, we carried out the first comprehensive mutation analyses ofthe NF1 gene in colorectal carcinomas in comparison with alterationsof BRAF, KRAS, and RASSF1A in a sample series selected to include acomparable number of samples with and without the microsatelliteinstability phenotype.samples displayed microsatellite instability (MSI), whereas 36 weremicrosatellite-stable (MSS). All tumors were nonfamilial as assessedby written questionnaires and cross check with the Norwegian CancerRegistry [17]. The colon, including the rectum, was divided intoproximal and distal sections: the proximal, or right side, spans fromcecum to two thirds of the way across transversum; the distal, or leftside, comprises the last third of the transversum, sigmoideum, andthe rectum. Of the 65 samples, 23 were located in the proximalcolon and 42 were located in the distal colon. The carcinomas arefrom a prospective series collected from seven hospitals in the Southeastregion of Norway during 1987–1989 and contain, on average,84% tumor cells [18]. The tumors have been selected to achievea consistently higher number of MSI-positive tumors compared tothe normal distribution (15%). By stratifying the samples accordingto the MSI status, we ensured that any results associated with theMSI or MSS group would be detected.NF1 Mutation Screening — DNA Amplificationand Denaturing High-Performance LiquidChromatography AnalysisTwenty-four representative CRC samples were analyzed for mutationsin the NF1 gene. These samples were selected to resemble theremaining series with regards to sex, age, tumor location, MSI status,and KRAS and BRAF mutation status. The 61 NF1 gene exons wereamplified in 61 polymerase chain reaction (PCR) fragments of 172to 579 bp. The primers were generally positioned approximately 50to 60 bp from the intron–exon boundary to allow the detection ofsplicing defects while minimizing intronic polymorphisms. In total,19,843 bases were screened per sample to obtain the final mutationstatus. The dHPLC was carried out as previously published[19], with minor alterations in the PCR protocol and denaturinghigh-performance liquid chromatography (dHPLC) methods. Fordetails concerning the dHPLC, please refer to Table W1.In short, the initial PCR was carried out in 25 μl of reaction volumesand was cooled at room temperature for 60 minutes to yieldheteroduplex formation. The identification of somatic NF1 gene mutationswas carried out with dHPLC on a 3500HT WAVE DNAfragment analysis system (Transgenomic, Crewe, UK) equipped witha DNASep column (Transgenomic). Polymerase chain reaction productswere examined through a 5% linear acetonitrile gradient forheteroduplexes with a separation flow rate of 1.5 ml/min. Commerciallyavailable WAVE Optimized Buffers (A, B, and D; Transgenomic)and Syringe Solution (Transgenomic) were used to provide highly reproducibleretention times with WAVE System instrumentation. Resolutiontemperatures and starting concentrations of buffer B fordHPLC analysis are reported in Table W1.Materials and MethodsTissue SpecimenSixty-five sporadic colorectal carcinomas from 64 patients with amean age of 70 years (range 33–92 years), and an equal distributionof male–female were included in the present study. Twenty-nine2 Sanger Institute — Catalogue of Somatic Mutations in Cancer (COSMIC) Web site.SequencingFor each dHPLC abnormal elution profile, genomic DNA wasreamplified with dHPLC primers and directly sequenced in both directionson a 3100 Genetic Analyzer (Applied Biosystems, Foster City,CA). Forward and reverse sequences were analyzed and compared withthe mRNA reference sequence and with the chromosome 17 genomiccontig reference sequence (NM_000267). The first base (position +1)of the initiator methionine is taken as the start of the cDNA. All missenseand splicing mutations detected were absent on 200 controlchromosomes belonging to the unaffected subjects.